Transthyretin (TTR) misfolding and amyloid aggregation is associated with a group of fatal disorders known as TTR amyloidosis (ATTR). Most of these diseases are hereditary, being caused by mutations that destabilize TTR's native state. Noteworthy, wild-type protein deposition also occurs and is considered a common cause of heart failure in the elderly. Small molecules that bind TTR can act as kinetic stabilizers, preventing tetramer dissociation and thus aggregation. In this context, our group repurposed tolcapone, an FDA-approved molecule for Parkinson¿s Disease, as a potent TTR aggregation inhibitor. Importantly, tolcapone¿s unique ability to cross the blood-brain barrier suggests that it could be an option for leptomeningeal amyloidosis, a rare type of ATTR that cannot be addressed with any of the current therapies. In the present thesis, we demonstrated that tolcapone binds to and inhibits the aggregation of TR variants associated with leptomeningeal amyloidosis, suggesting that it can become the first broad-spectrum drug to treat all manifestations of the disease. Despite the excellent performance of tolcapone, the structures of TTR:tolcapone complexes revealed that it could be redesigned to establish more contacts with the protein, which could improve its potency. By combining rational design and molecular dynamics simulations we discovered M-23, a molecule that presents one of the highest affinities for TTR reported thus far. The crystal structure confirmed that, as intended, M-23 forms new and strong contacts with the protein, leading to a higher tetramer stabilization both in vitro and in serum relative to tolcapone. These results encouraged us to further develop M-23 into a drug for ATTR, yet its poor pharmacokinetic profile could compromise its therapeutic use. In this context, we developed PITB, a M-23 derivative that keeps the interactions it establishes with TTR while improving its pharmacokinetics. The structural and biophysical characterization of PITB interaction with TTR revealed that it binds with high affinity to both the wild-type protein and the two most clinically significant TTR variants, stabilizes them, and inhibits their aggregation. Most importantly, PITB exerted a higher tetramer stabilizing effect in plasma from patients than tolcapone, which together with its remarkable pharmacokinetics make of PITB a very promising candidate to treat ATTR and at an affordable cost for all patients. While much insight has been gained in these studies through three-dimensional structures, they do not shed light into the dynamics of the conformational alterations involved in the amyloidogenicity of disease-related TTR variants or assess the effect of molecular binders. To address this gap, in this thesis we exploited the potential of two mass spectrometry-based footprinting methods, hydrogen deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP). The data obtained provided valuable insights into the effect of pathogenic mutations and kinetic stabilizers on TTR structure, opening a new avenue for the personalized screening of TTR kinetic stabilizers targeting specific variants. All in all, the results obtained in this thesis provide significant understanding of the structural-determinants of TTR kinetic stabilization, and support the development of the molecules presented here as drugs to treat ATTR.
Advancing in the treatment of transthyretin amyloidosis : Utilizing structure-driven approaches to develop kinetic stabilizers
Garcia de Carvalho Pinheiro, F. (Author). 18 Jul 2023
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis